2-amino-5, 6-dihydropteridines



United States Patent C 3,067,293 Z-AMlNO-S,-DIHYDRQITERIDINES Meyer Sletzinger, North Plaintield, and Max Tisiiler,

Westfield, NJ assignors to Merck & Co., Inc, Rahway, NJ a corporation of New .iersey N Drawing. Original appiieaticn May 3, 1954, Set. N11 427,331, now Patent No. 2,821,558, dated Jan. 23, 1958. Divided and this application Felt. 2t), 1957, Ser. No. 641,247

12 Claims. (ill. 260-2515) This invention relates to pteroylglutamic acid, commonly known as folic acid. More particularly, it is concerned with synthetic methods of producing pteroylglutamic acid and novel intermediates useful therein.

This application is a continuation-in-part of Serial No. 334,382, filed January 30, 1953 now abandoned, and a division of Serial No. 427,331, filed May 3, 1954, now United States Patent 2,821,558.

Pteroylglutamic acid, or vitamin B occurs naturally -in yeast, liver, grasses, and mushrooms. This substance has been found to be therapeutically efiective in the treat- .{ment of macrocytic anemias, sprue, and other conditions of the circulatory system. Although pteroylglutamic acid can be isolated from natural sources, it has been found that chemical synthesis of the vitamin is a more desirable method of production. However, the syntheses of pteroylglutamic acid reported heretofore have not been entirely satisfactory due to low yields and inherent difficulties caused by the very low solubility of the intermediates employed.

It is therefore an object of this invention to provide a new and novel synthesis of pteroylglutamic acid. Another object is to provide a process for producing pteroylglutamic acid from readily available, inexpensive starting materials. A further object is to provide a process for producing pteroylglutamic acid wherein crystalline, soluble intermediates are employed. It is also an object to provide new and novel intermediates useful in preparing pteroylglutamic acid. Other objects will be apparent from the following detailed description of the invention.

According to the present invention, it has been discovered that these objects and others can be achieved when pteroylglutamic acid is produced by the process which comprises reacting a 1,2-dihalo-1,3,3trisubstituted propane wherein the 1,3,3, substituents are alkoxy, aryloxy or aralkoxy radicals (I) with a weak base to produce the corresponding 2-halo-3,3-dialkoxy, diaryloxy, or diaralkoxy propionaldehyde (II), condensing the resulting propionaldehyde with a 2,4,5-triamino pyrimidine substituted in the 6 position with a hydroxy, alkoxy, aryloxy, or aralkoxy radical (III) to produce a Z-amino- 5,6-dihydr opteridine of the formula N i T HzN-kN N wherein R is an alkoxy, aryloxy, or aralkoxy radical and R is a hydroxy, alkoxy, aryloxy, or aralkoxy radical (IV), oxidizing the substituted Z-amino-5,6-dihydropteridine to the corresponding 2-amino pteridine (V), acylating the said substituted 2-amino pteridine to produce the corresponding 2-acylamino pteridine (VI) and subsequently hydrolyzing it to produce a 2-acylamino-4-hydrooxy-6-formylpteridine (VII), reacting the 2-acylamino- 4-hydroxy-6-formylpteridine with L-(-1)N-(p-aminobenzoyl) glutamic acid to produce the Schifls base N-{p- [(2 acylamino-4-hydroxy-6-pteridylmethylene) imino] benzoyl} glutamic acid (IX), reducing said Schiifs base to N -acyl-N -formyl pteroylglutamic acid (X), and hy- 3,067,200 Patented Dec. 4, 1962 2 drolyzing said compound to pteroylglutamic acid (XI). This sequence of reactions can be illustrated as follows:

wherein X is a halogen, R is an alkoxy, aryloxy, or aralkoxy radical, R is a hydroxy, alkoxy, aryloxy, or aralkoxy radical, and R is an acyl radical.

The starting materials which can be used in practicing this invention are the 1,2-dihalo-1,3,3-trialkoxy propanes, 1,Z-dihalo-1,3,3-triaryloxy propanes, and the 1,2- dihalo-l,3,3-triaralkoxy propanes. Such compounds are readily prepared by halogenating a 1,3,3-trialkoxy, triaryloxy, or triaralkoxy propylene-1 according to the method disclosed in the J. Am. Chem. Soc., 73, 206 (1951). For example, 1,2-dibromo-1,3,3,-triethoxy propane is produced by reacting 1,3,3-triethoxy propylene-1 with bromine in an inert solvent. In a similar manner, 1,2-dibromo-1,3,3-tribenzyloxy propane is prepared by reacting acrolein dibromide with benzyl alcohol in the presence of hydrochloric acid to form 2-bromo-1,3,3-tribenzyloxy propane, reacting said compound with an inorganic base in the presence of benzyl alcohol to produce 1,3,3-tribenzyloxy propylene-1 which is then readily brominated to the desired 1,Z-dibromo-1,3,3-tribenzyloxy propane. Other compounds which can be prepared according to this method by use of the corresponding alcohol and halogen are 1,2-dichloro-1,3,3,-trimethoxy propane, 1,2-dichloro-l,3,3-tributoxy propane, and 1,2-dibromo-l,3,3-tripropoxy propane. After the reaction has been terminated the 1,2-dihalo-1,3,3-trisubstituted propane can be isolated from the reaction mixture and purified or the reaction mixture can be used directly as the starting material in our process without further processing.

In the first step of the process for preparing pteroylglutamic acid according to the preferred method of this invention, a 2-halo-3,3-dialkoxy, diaryloxy, or diaralkoxy propionaldehyde is produced by reacting the correspondnig 1,Z-dihalo-1,3,3-trialkoxy, triaryloxy, or triaralkoxy propane with a weakly basic substance. Examples of weak bases which are suitable for effecting the reaction are sodium acetate, sodium bicarbonate, ammonium hydroxide, and the like. The reaction is conveniently conducted in the presence of a suitable inert solvent or mixture of solvents. In general, hydroxylated solvents such as the alcohols are not used as the reaction medium since such solvents are not completely inert under the conditions of reaction. Some examples of solvents which can be used are water, dioxane, ether, chloroform, carbon tetrachloride, acetone, benzene and formamide. The reaction proceeds satisfactorily within a wide range of temperature. Thus lowered temperatures of about C. and elevated temperatures such as 80 C. can be used with good results. The resulting 2-ha1o-3, 3-dialkoxy, diaryloxy, or diaralkoxy propionaldehyde can be recovered from the reaction mixture by conventional methods. Examples of the novel compounds which can be prepared by this process are 2-bromo-3,3-diethoxy propionaldehyde, 2-bromo-3,3-dibenzyloxy propionaldehyde, 2-chloro-3,3-dipropoxy propionaldehyde, and 2-bromo- 3,3-dibutoxy propionaldehyde.

The pteridine moiety, which is an essential structural unit of pteroylglutamic acid, is produced by condensing a 2-halo-3,3-dialkoxy, diaryloxy, or diaralkoxy propionaldehyde with a 2,4,5-triamino pyrimidine having in the 6 position a hydroxy, alkoxy, aryloxy, or aralkoxy radical to produce the corresponding 2-amino-6-dialkoxymethyl, diaryloxymethyl, or diaralkoxymethyl-S,6-dihydropteridine. Fortunately, the resulting pteridine moiety is practically all the 6-rnethyl position isomer needed in the synthesis of biologically active compounds such as pteroylglutamic acid. The condensation is preferably effected by intimately contacting the reactants in the presence of a solvent and a condensing agent. The solvent can be Water or an inert organic solvent or mixture of such solvents. Illustrative of solvents which can be used are hydroxylated solvents such as alcohols and glycols, particularly ethyl alcohol and ethylene glycol, and solvents such as acetone, benzene, and formamide. Examples of condensing agents which might be mentioned are sodium acetate, disodium phosphate, silver carbonate, and sodium formate. The reaction will proceed at ordinary temperatures but the rate of reaction Egg 8e increased by using elevated temperatures such as The condensation resulting in formation of the pteridine nucleus proceeds satisfactorily regardless of the substituent in the 6 position of the 2,4,5-triamino pyrimidine used as reactant. Thus, equally good results are obtained when the 6-substituent is a hydroxy, an alkoxy radical such as ethoxy, propoxy, or butoxy, aryloxy radical such as phenoxy, or an aralkoxy radical such as benzyloxy. However, the condensation is most easily accomplished when a 2-halo-3,3-dialkoxy, diaryloxy, or diaralkoxy propionaldehyde is used in which the halogen is bromine or chlorine. In specific embodiments of this condensation 2-amino-4-benzyloxy-6-diethoxymethyl- 5,6-dihydropteridine and 2-amino-4-hydroxy-6-diethoxymethyl-S,6-dihydropteridine can be produced by condensing respectively 2,4,5-triamino-6-benzyloxy pyrimidine and 2,4,5-triamino-6-hydroxy pyrimidine with 2- bromo-3,3-diethoxy propionaldehyde in the presence of aqueous ethanol and sodium acetate. After completion of the condensation the desired product can be recovered from the reaction mixture by conventional procedures or the reaction mixture can be used directly in the preparation of the fully aromatic pteridine moiety.

Alternatively, the 2-amino-6-dialkoxymethyl, diaryloxymethyl and diaralkoxymethyl-S,6-dihydropteridines may be conveniently produced by contacting directly a 2,4,5- triarnino pyrimidine having an alkoxy, aryloxy or aralkoxy substituent in the 6 position with a 1,2-dihalo-l,3,3 trisubstituted propane in the presence of a weak base and a suitable solvent. This reaction may be readily effected by the use of reactants and weak bases as previously described herein. Solvents such as water, ether, dioxane, acetone and the like may be used for the reaction. The reaction goes to completion in about onehalf hour to two hours. Once reaction is completed, the desired product can be recovered from the reaction mixture by conventional methods. In a specific illustration of this reaction 2,4,5-trian'1ino-6-hydroxy pyrimidine is reacted with 1,2-dibromo-1,3,3-triethoxy propane in the presence of a weak base to produce 2-amino-4-hydroxy- 6-diethoxyrnethyl-5,6-dihydropteridine. Similarly 1,2-dichloro-1,3,3-tribenzyloxy is contacted with 2,4,5-triamino- 6-1ydroxy pyrimidine and a weak base to form Z-amino- 4-hydroxy-6-dibenzyloxymethyl-5,6-dihydropteridine.

The fully aromatic Z-amino pteridines having a hydroxy, alkoxy, -aryloxy, or aralkoxy radicalin the 4 position and a dialkoxymethyl, diaryloxymethyl, or di- =aralkoxymethyl radical in the 6 position can be prepared by dehydrogenation of the correspondingly substituted 5,6-dihydropteridines. The dehydrogenation is readily accomplished by intimately contacting the 5,6-dihydropteridine with a mild oxidizing agent. Specific examples of suitable oxidizing agents are air, oxygen, iodine, and hydrogen peroxide with an iron catalyst. In general, it is preferable to maintain a pH of about 8 to 9 to obtain best results. In addition, the reaction is conveniently accomplished in a suitable inert solvent such as alcohols, glycols, acetone, benzene, formamide, dioxane, and water. The resulting pteridines can be isolated from the reaction mixture by conventional methods. According to specific applications of this dehydrogenation reaction Z-amino- 4-benzyloxy-6-diethoxymethyl pteridine and 2-amino-4- hydroxy-6-diethoxymethyl pteridine are produced by oxidizing respectively 2-amino-4-benzy1oxy-6-diethoxymethyl-5,6-dihydropteridine and 2-amino-4-hydroxy-6-di ethoxy-methyl-S,6-dihydropteridine with hydrogen peroxide and ferrous sulfate in a suitable solvent. Examples of other representative pteridines which can be prepared in this manner are 2-amino-4-butoxy-6-dibenzyloxymethyl pteridine, 2-amino-4-ethoxy-6-dimethoxymethyl pteridine,

and Z-amino-4-methoxy-6-dipropoxymethyl pteridine.

In the next step of the process Z-amino pteridines containing a hydroxy, alkoxy, aryloxy, or aralkoxy substituent in the 4 position and a dialkoxymethyl, diaryloxymethyl, or diaralkoxymethyl substituent in the 6 position are converted to the corresponding novel 2-acylamino pteridines. The acylation can be effected by intimately con tacting the substituted Z-amino pteridines with a suitable acylating agent such as an acyl halide or carboxylic acid anhydride. Acetyl chloride, propionyl chloride, butyryl chloride, benzoyl bromide, acetic anhydride, propionic anhydride, butyric anhydride, and benzoic anhydride are examples of suitable acylating agents. The acylation is conducted in a liquid reaction medium which can be an inert organic solvent or an excess of the acylating agent. In general, an added solvent is not required since the acylating agents are usually liquids at normal or slightly elevated temperatures. Although the reaction proceeds at ordinary temperatures it is usually affected at higher temperatures such as the reflux temperature to enhance the rate of reaction. The desired 2-acylamino pteridine can be isolated from the reaction mixture by conventional methods such as cooling and filtering to separate the crystalline product. According to this acylation procedure, Z-acylamnio pteridines having the described substituents in the 4 and 6 positions can be readily prepared wherein the acyl substituent is an alkyl, aryl, or aralkyl carbonyl radical. Thus, some specific Z-acylamino pteridines which can be produced according to this process are 2-propionamido 4 benzyloxy 6 dimethoxymethyl pteridine, 2 butyramido-4-hydroxy 6 dipropoxymethyl pteridine, 2-acetamido-4-phenoxy-6-diethoxymethyl pteridine, Z-acetamido-4-hydroxy-6-diethoxymethyl pteridine, 2-benzamido-4-hydroxy-6-diethoxymethyl pteridine, and 2-phenylacetamido-4-hydroxy-6-diethoxymethyl pteridine.

The acylated pteridines possess unique and valuable properties which distinguish them from the non-acylated pteridines. For example, the non-acyiated pteridines reported in the art are amorphous compounds which are nearly insoluble in ordinary solvents. Therefore, it was indeed surprising to discover that acylated pteridines, and derivatives of acylated pteridines, could be readily produced in crystalline form. Furthermore, the acylated pteridines were found to have an unexpectedly high solubility in water and many organic solvents. The ability to produce crystalline compounds with high solubility by the introduction of an acyl group on the 2-amino was entirely unexpected since pteridines having such desirable properties were heretofore unknown. This combination of desirable properties greatly enhances the usefulness of the 2-acylamino pteridines. Thus, the production of crystalline pteridines is a great aid in the purification of such compounds. Because of their greater solubility, the Z-acylamino pteridines can be used in reactions with smaller volumes of solvents than the non-acylated compounds thereby allowing a saving in material and permitting greater manipulative freedom.

In the next step of the process, 2-acylamino pteridines substituted in the 4 position with a hydroxy, alkoxy, aryloxy, or aralkoxy radical and in the 6 position with a dialkoxymethyl, diaryloxymethyl, or diaralkoxymethyl radical are hydrolyzed to the corresponding 2-acylamino- 4-hydroxy-6-formyl pteridine. According to this hydrolysis reaction the acetal in the 6 position is converted to a formyl radical. Simultaneously pteridines which contain an alkoxy, aryloxy, or aralkoxy radical in the 4 position are hydrolyzed to the corresponding 4 hydroxy pteridines. Either mineral or organic acids can be used for the hydrolysis. Examples of some suitable acids are hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, and formic acid. The reaction is readily conducted in a solvent medium which can be an excess of the acid used or an added solvent such as water or an inert organic solvent. Normal or somewhat elevated temperatures may be used to promote the reaction. After the hydrolysis has been completed crystalline 2-acylamino-4-hydroxy-6- formyl pteridine is isolated by conventional methods. In specific applications of this reaction 2-acetamido-4-hydroxy-6-diethoxymethyl pteridine and 2-propionamido-4- benzyloxy-6-dimethoxymethyl pteridine are hydrolyzed with formic acid to 2-acetamido-4-hydroxy-6-formyl pteridine and 2-propionamido-4-hydroxy-6-formyl pteridine. Other similar compounds which can be prepared in this manner are 2-benzamido-4-hydroxy-6-formyl pteridine, 2-butyramido-4-hydroxy-6-formyl pteridine and 2- phenylacetamido-4-hydroxy-6-formyl pteridine.

In the succeeding step the Schiffs base N-{p-[(2-acylamino 4 hydroxy 6 pteridylrnethylene)imino]benzoyl} glutamic acid is produced by contacting the 2-acylamino-4-hydroxy-6-formyl pteridines with L(-1)N-(paminobenzoyl) glutamic acid in the presence of a solvent. In general, insert organic solvents can be used for the reaction medium. Some examples of suitable solvents are the alcohols such as methanol, ethanol, propanol and dioxane. The reaction can be carried out at room temperature or more elevated temperatures. By cooling the mixture after the reaction has been completed the desired Schiffs base crystallizes from solution and is recovered by conventional methods. According to this procedure N {p [(2 acylamino 4 hydroxy 6 pteridylmethylene)imino]benzoyl} glutamic acids can be prepared wherein the acyl group is an alkyl carbonyl, aryl carbonyl or aralkyl carbonyl radical by selecting the corresponding 2-acylamino-4-hydroxy-6-f0rmyl pteridines for the reaction. Representative of the broad class of Schiffs bases which can be prepared by this process are N-{p-[(2- acetamido 4 hydroxy 6 pteridylmethylene)imino1- benzoyl} glutamic acid, N-{p-[(2-benzamido-4-hydroxy- 6-pteridylmethylene)imino1benzoyl} glutamic acid and N {p [(2 butyramido 4 hydroxy 6 pteridylmethylene)imino]benzoyl} glutamic acid. I

According to the next step of this process N -acyl-N formyl pteroylglutamic acid is produced wherein the acyl' substituent is an alkyl carbonyl, aryl carbonyl or aralkyl carbonyl radical by contacting the N-{p[(2-acylamino-4- hydroxy 6 pteridylmethylene)imino]benzoyl} glutamic acid with formic acid. The reaction is conveniently conducted in the presence of a solvent. Examples of suitable solvents are the hydroxylated solvents such as the lower alcohols, dioxane, ether and the inert chlorinated hydro carbons. In addition, an excess of formic acid may be used for the reaction medium. A small amount of acetic anhydride is ordinarily added in effecting the reactionsince it serves to promote high yields of the desired N acyl-N -formyl pteroylglutamic acids. Although the reaction proceeds at ordinary temperatures the rate of reaction is increased at elevated temperatures. After'the reaction has been completed the desired product can be isolated by cooling the reaction mixture and adding ether to precipitate the product. In one embodiment of this method N -acetyl-N formyl pteroylglutamic acid is produced by reacting N-{p-[(2-acetamido-4-hydroxy-6-pteridylmethylene)imino]benzoyl} glutamic acid in dioxane with formic acid. Representative of other compounds which can be prepared according to this reaction by employing the appropriate Schifis base are N -benzoyl-N formyl pteroylglutamic acid, N butyryl N formyl pteroylglutamic acid, N -propionyl-N -forrnyl pteroylglutamic acid and N -phenylacetyl-N -formyl pteroylglutamic acid.

According to the next and last step, pteroylglutamic acid is produced by hydrolyzing N -acyl-N -formyl pteroylglutamic acid with an inorganic or organic acid or base. It is preferred, however, to employ the common inorganic acids and bases such as sodium hydroxide, potassium hydroxide, hydrochloric acid, sulfuric acid, and phosphoric acid. The reaction is run by intimately contacting the reactants in a suitable solvent such as water or an inert organic solvent. Elevated temperatures can be used, if desired, to promote the reaction. After the hydrolysis has been completed the pteroylglutamic acid' is isolated from the reaction mixture by conventional metheds.

The examples which follow illustrate methods of carrying out of the present invention but it is understood, however, that these examples are not to he considered as limiting the invention.

EXAMPLE 1 Production of 2-Bromo-3,3-Diethoxy-Propionaldehyde 1,2-dibro1no-1,3,3-triethoxy propane was added to a solution of 27.6 gm. of sodium bicarbonate in a mixture of 30 ml. of water and ml. of dioxane at a temperature below 10 C. After all the 1,2-dibromo-1,3,3-triethoxy propane was added, stirring was continued for 2 hours. The reaction mixture was extracted with ethyl ether and the ether extract washed with water and dried over anhydrous sodium sulfate. The ether solution was filtered and concentrated on a steam bath under reduced pressure to remove the solvent. The resulting liquid was fractionated through a distillation column. The yield was 26 gm. of 2-bromo-3,3-diethoxy propionaldehyde hav- :7 ing a boiling point of 6365 C. at 2.5 mm. of mercury; 71 C.=l.4513.

The 1,2-dibrorno-1,3,3-triethoxy propane used as the starting material was prepared by reacting 50 gm. of 1,3,3-triethoxy-propylene-1 in 125 ml. of ether with 41 gm. of bromine while stirring and maintaining the temperature between 10 C. The solution was stirred for 1 hour at 0-10 C. and then the solvent removed under reduced pressure to produce a residue of 1,2-dibromo- 1,3,3-triethoxy propane.

EXAMPLE 2 Production of 2-Chl0r0-3,3-Diethoxy- Propionaldehyde To a 100 ml. 3-necked flask equipped with stirrer, thermometer, and gas inlet was added 34.8 gm. of 1,3,3- triethoxy-propylene-l. It was cooled with stirring to about C. and then 13.6 gm. of chlorine was bubbled through the liquid over a period of 45 minutes. The reaction mixture containing 1,2-dichloro-1,3,3-triethoxy propane was then added under nitrogen and with stirring to a mixture of 21 ml. of water, 62.5 ml. of dioxane, and 26.4 gm. of sodium bicarbonate. The reaction mixture was maintained at 510 C. and stirred for 2 hours. The reaction mixture was extracted twice with 50 ml. portions of ether. The combined ether extracts were washed with water and dried over sodium sulfate. The ether solution was filtered and removed under reduced pressure. The residue was distilled over calcium carbonate under reduced pressure to give 2-chloro-3,3- diethoxy-propionaldehyde having a boiling point of 58- 60 C. at 1.2 mm. of mercury, n C.=1.4309.

In the same manner, 2-chloro-3,3-dipropoxy-propionaldheyde, 2-chloro-3,3-dibenzyloxy-propionaldehyde and 2-chloro-3,3-dibutoxy propionaldehyde can be prepared by employing the corresponding 1,2-dichloro substituted propane.

EXAMPLE 3 Production of Z-Amino-4-Benzyloxy-6-Diethoxymethyl- 5,6-dihydropteridine To a solution of 5 gm. of 2-bromo-3,3-diethoxy-propionaldehyde in 70 ml. of ethanol was added a solution of 2,4,5-triamino-6-benzyloxy pyrimidine in 70 ml. of ethanol containing 2 gm. of sodium acetate. The solution was stirred at room temperature for 15 minutes and subsequently heated at 60-65 C. for 1 /2 hours. The reaction mixture was cooled to room temperature and added to 700 ml. of water with stirring to prepare for purification of the product.

To the resulting slurry 2.5 N hydrochloric acid was added until the mixture became acidic. Insoluble matter that formed was removed by filtration. After cooling, the filtrate was added to an excess of 6 N ammonium hydroxide at a temperature of 5-10 C. The amorphous precipitate was removed by filtration and dried. The yield of 2-amino-4-benzyloxy-6-diethoxymethyl 5,6 dihydropteridine was 3.5 gm.

A sample was purified further by dissolving it in ethyl acetate and adding n-hexane until the product precipitated.

EXAMPLE 4 Production of Z-Amino-4-Benzyloxy-6- Diethroxymethyl Pteridine To an ethanol solution of 2-amino-4-benzyloxy-6-diethoxymethyl-S,6-dihydropteridine was added 50 mg. of ferrous sulfate in 1 ml. of water and then 2.3 gm. of 30% hydrogen peroxide in 10 ml. of water was added over a 30 minute period. The mixture was concentrated under reduced pressure to a small volume and 2.5 N hydrochloric acid added to dissolve most of the oil. The solution was separated from insoluble material by decantation and added to an excess of cold 6 N ammonium hydroxide. A precipitate resulted which was g. isolated and dried. The yield of 2-amino-4-benzyloxy-6- diethoxymethyl pteridine was 4 gm. and its ultraviolet absorption spectrum in 0.1 N NaOH had maxima at 2560 A. (E%:369) and 3610 A. (E%=210); in 0.1 N HCl it had a maximum at 3350 A. (E%=364).

By a paper strip chromatography, run according to the procedure of Weygand in Experientis 6, 184 (1950), of the acid obtained by permanganate oxidation of 2- arnino-4-hydroxy-6-formyl pteridine which was produced by acetal hydrolysis of 2-amino-4-benzyloxy-6-diethoxymethyl pteridine. It was found that the product was all the desired position 6 isomer.

The solution of 2-amino-4-benzyloxy-6-diethoxymethyl-5,6-dihydropteridine used in this example was prepared by reacting 4.6 gm. of 2,4,5-triamino-6-benzyloxy pyrimidine and 1.8 gm. of sodium acetate in 63 ml. of ethanol with 4.5 gm. of 2-bromo-3,3-diethoxy-propionaldehyde in 63 ml. of ethanol.

EXAMPLE 5 Production of 2-Amino-4-Hydr0xy 6 Diethoxymethyl- 5,6-Dihydropteridine and 2-Amin0-4-Hydr0xy 6 Diethoxymethyl Pteridine 5.0 gm. of 2,4,5-triamino-6-hydroxy pyrimidine sulfate was dissolved in ml. of water containing 5 gm. of barium chloride. The solution was heated to 60 C. in a nitrogen atmosphere for 1 hour with stirring and subsequently filtered hot to remove the insoluble precipitate.

To the resulting solution of 2,4,5-triamino-6-hydroxy pyrimidine was added 140 ml. of ethanol and then 5.14 gm. of 2-bromo-3,3-diethoxy-propionaldehyde. The reaction mixture was stirred under nitrogen at room temperature for 56 hours to yield a solution of 2-amino-4- hydroxy-6 diethoxymethyl 5,6 dihydropteridine. The solution was then adjusted to pH 89 and 50 mg. of ferrous chloride and 2.6 gm. of 30% hydrogen peroxide added at room temperature. The solution was stirred 5 hours, filtered and the precipitate washed with water, alcohol, and ether to yield purified 2-amino-4-hydroxy-6- diethoxymethyl pteridine.

A sample was purified by conversion to the sodium salt and reprecipitation of the original free base. It had an ultraviolet absorption curve which exhibited maxima in 0.1 N HCl at 3180 A. (E%=340) and in 0.1 N NaOH at 2550 A. (E% =941), 3620 A. (E% :288).

In a similar manner, 2-amino-4-hydroxy-6-diethoxymethyl pteridine was prepared by reacting 2-chloro-3,3- diethoxy-propionaldehyde with 2,4,5-triamino-6-hydroxy pyrimidine in ethanol and in the presence of sodium acetate to produce 2-amino-4-hydroxy-6-diethoxymethyl-5,6- dihydro-pteridine which was oxidized with hydrogen peroxide and ferrous sulfate to 2-amino-4-hydroxy-6-diethoxymethyl pteridine.

EXAMPLE 6 Production of 2-Amino-4-Hydroxy-6-Dibenzyloxymethyl- 5,6-Dihydropteridine and 2-Amino-4-Hydroxy-6-Dibenzyloxymethyl Pteridine After passing nitrogen through a stirred solution of 8.2 gm. of sodium acetate in a mixture of 200 ml. of ethanol for one-half hour, 4.7 gm. of 2,4,5-triamino-6-hydroxy pyrimidine dihydrochloride and 7.0 gm. of 2-bromo-3,3- dibenzyloxy-propionaldehyde were added to the solution. The reaction mixture was stirred overnight at room tem perature, forming Z-amino 4 hydroxy-6-dibenzyloxymethyl-5,6-dihydropteridine in solution. About 0.1 gm. of ferrous sulfate was dissolved in 3 ml. of water and added to the reaction mixture together with 30 gm. of 10% hydrogen peroxide added dropwise. The reaction mixture was filtered after standing overnight to give 2- amino-4-hydroxy-6-dibenzyloxymethyl pteridine which is readily purified by preparation of its sodium salt and reprecipitation of the free base.

EXAMPLE 7 To a solution of 30 gm. of 2,4,5-triamino-6-hydroxy pyrimidine in 422 ml. of water and 53 gm. of sodium bicarbonate is added over a 30 minute period a solution of 1,Z-dibromo-1,3,3=triethoxy propane prepared as hereinafter described. After the addition was completed the solution was stirred for 2hours. To the reaction mixture containing, 2-amino-4-hydroxy-6-diethoxymethyl-5,6-dihydropteridine was added 45 gm. of hydrogen peroxide in 90' ml. of water. After filtering the mixture to obtain the precipitated 2 amino-4-hydroxy-6-diethoxymethyl pteridine it was washed with water, acetone and ether.

The solution of 1,Z-dibromo-1,3,3-triethoxy propane used in this example was prepared by adding 22.6 gm. of 1,1,3-triethoxy propane to 44 ml. of anhydrous ether and then 19.5 g. of bromine while stirring and cooling to -10 C. to C.

The'method of this example is followed and 1,2-dichloro-l,3,3-tribenzyloxy propane is contacted with 2,4,5- triamino-6-hydroxy pyrimidine and sodium bicarbonate to form 2-amino-4-hydroxy-6-dibenzyloxymethyl-5,6-dihydropteridine which, upon oxidation with hydrogen peroxide, forms 2-amino-4-hydroxy-6-dibenzyloxymethyl pteridine.

EXAMPLE 8 Production of 2-Acetamido-4-Hydroxy-6- Diethoxymethyl Pteridine To a 3-necked flask equipped with stirrer and refl condenser was added 4.2 gm. of 2-amino-4-hydroxy-6- diethoxymethyl pteridine and 80 ml. of acetic anhydride. The mixture was refluxed with stirring for 1.5 hours. To the solution was added 2 gm. of activated carbon and refluxing was continued for 10 minutes. The hot solution was filtered and cooled in the ice-box overnight. The crystalline precipitate which formed was filtered and washed with 10 ml. of cold acetic anhydride and then with ether. The precipitate was dried at 50 C. under reduced pressure to yield white crystalline 2-acetamido-4- hydroxy-6-diethoxymethyl pteridine. A 1 gm. sample of the product was recrystallized from 2 ml. of dioxane and melted at 198-200 C.

The ultraviolet absorption curve exhibited maxima in 0.1 N NaOH at 2560 A. (E%=850) and 3500 A. (E%=240).

EXAMPLE9 Production of Z-Propionamido 4-Hydroxy-6- Diethoxymethyl Pteridine A slurry of 3.5 gm. of 2-amino-4-hydroxy-6-diethoxymethyl pteridine in 70 gm. of propionic anhydride was heated at 140 C. for five hours during which time solution was eflected. To the reaction mixture was added 1.5 gm. of charcoal and the mixture filtered. The filtrate was evaporated to about one-half volume and allowed to stand overnight in the cold. The solid was collected by filtration, washed twice with ethyl ether and twice with petroleum ether. After drying in air the light tan crystals of 2-propionamido-4-hydroxy-6-diethoxymethyl pteridine weighed 2.95 gm.

EXAMPLE 10 Production of 2-Acetomido-4-Hydroxy-6- F ormyl Pteridine To a flask equipped with a stirrer was added 860 ml. of 98% formic acid and 58 gm. of 2-acetamido-4-hydroxy-6- diethoxymethyl pteridine. Complete solution was achieved in minutes. Upon standing for 15 minutes at room temperature a precipitate appeared. The solution was allowed to stand under nitrogen in an ice-box overnight. -It was filtered and the precipitate was washed with cold formic acid and then anhydrous ether giving. a.

formic acid salt of Z-acetamido-4-hydroxy-6-formyl pteridine.

The ultraviolet absorption curve exhibited maxima in 0.1 N NaOH at 2550 (E%=809) and 3500 A. (E% :294). The salt loses formic acid when heated at C. for three hours at 3 mm. pressure.

EXAMPLE 11 Production of N-{p-[(2-Acetamido-4-Hydroxy-6-Pteridyl- Methylene) Imin0]Benz0yl}Glutamic Acid To a 1 liter flask equipped with stirrer and reflux condenser containing 660 ml. of ethanol was added 6.6 gm. of L(-1)N-(p-aminobenzoyl) glutamic acid. The mixture was-refluxed in a nitrogen atmosphere until solution was complete (20 minutes). At this point 5.3 gm. of 2-acetamido-4-hydroxy-6-formyl pteridine was added.

Refluxing and stirring were continued for 1 hour during.

EXAMPLE 12 Production of N -Acctyl-N -Formyl Pteroylglutamic Acid A mixture of 100 ml. of 98% formic acid and 11.2 gm. of acetic anhydride was prepared and allowed to stand overnight. To 1 gm. of N-{p-[(2-acetamido-4-hydroxy-fi-pteridylmethylene) imino]benzoyl} glutamic acid was added 18 ml. of the formic acid-acetic anhydride mixture. The resulting yellow solution was heated at 67 C. for 1 hour during which time the solution became reddish. The solution was cooled to room temperature and combined with 200 ml. of anhydrous ether. A reddish precipitate formed which was filtered and washed well with ether without exposing it to the atmosphere. The resulting N -acetyl-N -formyl pteroylglutamic acid was dried under reduced pressure.

Various changes and modifications of the invention can be made and, to the extent that such variations incorporate the spirit of this invention, they are intended to be included within the scope of the appended claims.

What is claimed is:

1. A 2-amino-4-R-6-di R methyl-5,6 dihydropteridine, wherein R represents a member selected from the class consisting of lower alkoxy and 'benzyloxy radicals and R represents a member selected from the class consisting of hydroxy, lower alkoxy, and benzyloxy radicals.

2. 2-Amino-4-hydroxy-6-di lower alkoxymethyl-5,6-dihydropteridine.

3. 2-Amino-4-hydroxy-6-diethoxymethy1 5,6-dihydropteridine.

4. Z-Amino 4-benzyloxy-6-diethoxymethyl-5,6-dihydropteridine.

5. 2-Amino dropteridine.

6. The method of producing 2-amino-4-R'-6-di R methyl-5,6-dihydropteridine which comprises reacting 2- R"-3,3-di R propionaldehyde with 2,4,5-triamino-6-R'- pyrimidine in the presence of an inert solvent, wherein R represents a member from the group consisting of lower alkoxy and benzyloxy radicals, R is a member from the group consisting of hydroxy and 'benzyloxy, and R" is a halogen from the group consisting of chlorine and bromine.

7. The method of producing a 2-amino-4-hydroxy'6- di lower alkoxymethyl-S,6-dihydropteridine which com- 4-hydroxy-6-dibenzyloxymethy1-5,6-dihyprises reacting a 2-bromo-3,3-di-lower alkoxy propionaldehyde with 2,4,5-triamino-6-hydroxy pyrimidine in the presence of an inert solvent.

8. The method of producing 2-arnino-4-benzyloXy-6- diethoxyrnethyl-5,6-dihydropteridine which comprises reacting 2-bromo-3,3-dieth0xy propionaldehyde with 2,4,5- triamino-6-benzyloxy pyrimidine in the presence of an inert solvent.

9. The method of producing 2-amino-4-hydroXy-6-diethoxymethyl-S,6-dihydropteridine which comprises reacting 2-bromo-3,3-diethoxy propionaldehyde with 2,4,5- triamino-6-hydroxy pyrimidine in the presence of an inert solvent.

10. The method which comprises reacting a 1,2-di-R"- 1,3,3-tri R propane with a weak base in the presence of an inert solvent to form 2-R"-3,3-di R propionaldehyde and condensing said aldehyde with a 2,4,5-triamino-6-R- pyrimidine in the presence of an inert solvent to produce a 2-amino-4-R'-6 di R methyl-5,G-dihydropteridine, wherein R represents a member from the group consisting of lower alkoXy and benzyloxy, R is a member from the group consisting of hydroxy and benzyloxy, and R" is 5 imidine and a weak base in the presence of an inert solvent under reaction conditions to produce a 2-amino-4- R'-6-di R methyl-5,6-dihydropteridine wherein R represents a member from the group consisting of lower alkoxy and benzyloxy, R is a member from the group consisting 10 of hydroxy and benzyloxy, and R" is a halogen from the group consisting of chlorine and bromine.

12. The method which comprises contacting 1,2-dibromo-1,3,3-triethoxy propane with 2,4,5-triamino-6-hydroxy pyrimidine and a weak base in an inert solvent under re- 15 action conditions to produce 2-amino-4-hydroxy-6-diethoxymethyl-5,6-dihydropteridine.

References (Jited in the file of this patent UNITED STATES PATENTS Waller et al. Mar. 14, 1950 Geraci Mar. 21, 1950 

1. A 2-AMINO-4-R''-6-DI R METHYL-5,6 DIHYDROPTERIDINE, WHEREIN R REPRESENTS A MEMBER SELECTED FROM THE CLASS CONSISTING OF LOWER ALKOXY AND BENZYLOXY RADICALS AND R'' REPRESENTS A MEMBER SELECTED FROM THE CLASS CONSISTING OF HYDROXY, LOWER ALKOXY, AND BENZYLOXY RADICALS. 